Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

Abstract

A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C 4 F 5 ). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K d ‫؍‬ 3 ؋ 10 ؊10 M) when compared with the wild-type (3-C 4 F 5 ) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 Å) and without testosterone (2.10 Å) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.Steroid hormones have remained a great challenge for immunodiagnostics. They are small, rigid, hydrophobic molecules with only a few functional groups capable of specific interactions with antibodies. The number of different closely related steroids in human serum is high, their in vivo concentrations are low (down to a picomolar level), and their relative concentrations can vary greatly, even between normal healthy individuals. Furthermore, steroids are poorly immunogenic in mice and rats; two species from which monoclonal antibodies are usually generated, making it extremely difficult to produce high affinity and specificity anti-steroid monoclonal antibodies. In most diagnostic immunoassays of steroid hormones, rabbit polyclonal antibodies are used, despite the many drawbacks associated with the use of antisera. The supply of good polyclonal antibody reagents of uniform quality is a severe problem for the immunodiagnostic industry and requires continuous immunization of many laboratory animals.Antibody engineering provides excellent tools to tailor the properties of antibodies with respect to affinity, specificity, and performance for different applications. An in vitro process of antigen-driven selection, based on the display of antibody fragments on the surface of a filamentous bacteriophage, has been shown to be a powerful method for the selection of specific or improved...

Affinity maturation of antibodies assisted by in silico modeling

“…Moreover, it is not guaranteed that substituting any of the contact residues will improve binding. For example, randomization and selection studies often yield substituted residues that are not in contact with the antigen (17). In vitro affinity maturation by somatic hypermutation yields, as a rule, mutations that are located in the periphery of the paratope (18).…”

Recombinant Antibodies and Non-Antibody Scaffolds for Immunoassays

“…The CDRs of VH and VL are usually targeted for both haptens and protein antigens (Siegel, et al, 2008), and mutations in CDRs as opposed to within the framework residues generally contribute more to increases in affinity (Orcutt et al, 2011;Short et al, 2002). In one study, the most significant increase in affinity was correlated with mutations in the light chain CDR1 even though this CDR1 was not contacting the hapten directly (Valjakka et al, 2002). Hapten-specific antibodies whose affinity has been increased by site directed mutagenesis are listed in Table 4.…”

Improving Immunoassay Performance by Antibody Engineering

“…Thus, antibody affinity can be improved by in vitro maturation, i.e., mutation of the complementaritydetermining regions of scFv or Fab fragments. A substantial increase in affinity has been achieved by this approach (11 ), but the reported affinities have not been higher than those of the best native monoclonal antibodies. Another approach is to prepare monoclonal antibodies in animals other than mice and rats, which are closely related and produce similar antibody responses.…”

“…This is possibly already the case: assay manufacturers seldom disclose the characteristics of their reagents. The various steps used to produce scFv are well known, and other, even more sophisticated tricks for improving the properties of more or less artificial binders have been described (11)(12)(13). Thus, antibody affinity can be improved by in vitro maturation, i.e., mutation of the complementaritydetermining regions of scFv or Fab fragments.…”